Splice variants of CD40-receptor

ABSTRACT

The present invention concerns six novel variants of alternative splicing of the CD40 receptor.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/IL00/00427 which has an Internationalfilinq date of Jul. 19, 2000, which designated the United States ofAmerica and was published in English.

FIELD OF THE INVENTION

The present invention concerns novel nucleic acid sequences, vectors andhost cells containing them, amino acid sequences encoded by saidsequences, and antibodies reactive with said amino acid sequences, aswell as pharmaceutical compositions comprising any of the above. Thepresent invention further concerns methods for detecting nucleic acidand amino acid sequences in a sample.

BACKGROUND OF THE INVENTION

CD40 is a cell surface receptor, which belongs to the TNF-R family, andwhich was first identified and functionally characterized on Blymphocytes. However, in recent years it has become clear that CD40 ismore widely expressed, including expression on monocytes, dendriticcells, endothelial cells and epithelial cells. Therefore it is nowthought that CD40 plays a more general role in immune regulation.

The CD40 protein is a 45–50 kDa glycoprotein of 277 AA, the 193 AAextracellular domain is composed of four imperfect repeats of about 40residues, anchored by a superimposable pattern of six cysteines. Thisorganization is found in the other members of the TNF-R family.

CD40 is expressed by multiple cell types. In the hematopoietic system,it is expressed on CD45 hematopoietic progenitors, B cell progenitors,mature B lymphocytes, plasma cells, monocytes, dendritic cells,cosinophils, basophils and on some T lymphocytes. CD40 is also expressedon non-hematopoietic cells such as endothelial cells, fibroblasts andepithelial cells.

Expression cloning using a CD40-Fc fusion protein allowed the isolationof a CD40-ligand (CD40-L) from activated T cells. The human CD40-L is apolypeptide of 261 AA including a 215 AA extracellular domain with fivecysteins. CD40-L is a member of the Tumor Necrosis Factor family.

CD40-R bound to its ligand activates protein-tyrosine kinases, includingLyn and Syk and induces the tyrosine phosphorylation of multiplesubstrates including phosphatidylinositol 3-kinase and phospholipaseCγ2.

CD40 also interacts with TRAF3 protein (TNF-R Associated Factor-3), alsoidentified under the name CRAF1, CD40 bp, LAP1 and CAP1 (18-21). TRAF3is a 62 kD intracellular protein which is expressed in almost all celltypes. The protein contains several functional domains which might beinvolved in signal transduction.

In addition, CD40 has been demonstrated to associate with TRAF2, amolecule which also associates with TNF-R2. The induction of NF-_(κ)Bactivation via CD40 cross-linking (and also via TNF-R2) could beattributed to TRAF2 signaling.

CD40 ligation activates resting B cells as shown by increase in size andexpression of new surface molecules involved in homotypic andheterotypic aggregation (CD23, VLA-4), as well as T cell costimulation(CD80/CD86). Furthermore, CD40-activated B cells secrete a panel ofcytokines which may act as autocrine and paracrine growth anddifferentiation factors.

CD40 is further known to be expressed on professional antigen presentingcells such as monocytes and dendritic cells, and its ligation to theabove cells results in secretion of multiple proteins includingcytokines such as IL1, IL5, IL8 IL10, IL12, TNF_(α), MiPl_(α), as wellas enzymes such as matrix metalloproteinase (MMP).

The importance of this receptor-ligand pair for the cellular immuneresponse, has been demonstrated by the diminished immunity againstseveral pathogens in CD40 and CD40-L knockout mice. In keeping withthis, CD40 ligation turns on monocyte tumoricidal activity as well as NOsynthesis.

Administration of antibodies to CD40-L has been shown to prevent theestablishment of autoimmune symptoms in various murine models including:(1) collagen type II-induced arthritis: a model for human rheumatoidarthritis; (2) lupus nephritis in lupus prone mice that represent modelsfor the systemic lupus erythematosus; (3) protcolipoprotein inducedexperimental encephalomyelitis: a model of human multiple sclerosis.

Administration of anti-CD40-L antibodies further has been demonstratedto prevent the development of graft versus host disease (GVHD) thatoccurs as a major complication of allogeneic bone marrow transplantation(van Kooten, C. and Banchereau, J., Frontiers in Bioscience, d1-11 Jan.1, (1997)).

GLOSSARY

In the following description and claims use will be made, at times, witha variety of terms, and the meaning of such terms as they should beconstrued in accordance with the invention is as follows:

“CD40R Variants nucleic acid sequences”—the sequence shown in any one ofSEQ ID NO: 1 to SEQ ID NO: 6 sequences having at least 90% identity (seebelow) to said sequences and fragments (see below) of the abovesequences of least 20 b.p. long. These sequences are sequences codingfor a novel naturally occurring, alternative splice variants of thenative and known CD40R, depicted in the Swiss Prot as CD40_(—)HUMANunder Accession Number P25942 which is the sequence coding for the human45–50 kDa glycoprotein of 277 amino acid. It should be emphasized thatthe novel variants of the present invention are naturally occurringsequences resulting from alternative splicing of the CD40R gene and notmerely truncated, mutated or fragmented form of the gene.

The description of the CD40R variants and their difference from theoriginal sequence is summarized in Table 1 as follows:

TABLE 1 SEQ Length Description of ID New variant internal (amino thevariation (comparing to NO: name acids) the original sequences 1 160 Hasthe extracellular domain of the native CD40 gene from signal peptide upto the position of the amino acid before the transmembrane region. 2 246Has the extracellular domain of the native CD40 gene from signal peptideup to the position of the amino acid before the transmembrane region. 3Mcd40-pFB1-1-5 156 Contains 135 N-terminal amino acids, including entireTNFR-Cys repeats 1 and 2, and part of the TNFR-Cys repeat 3. Alternative21 amino acids at the C terminus. Missing the TNFR-Cys repeat #4, partof the TNFR-Cys repeat #3, the TM domain and the cytoplasmic domain 4MCD40Alt8 203 Contains 165 N-terminal amino acids, including entireTNFR-Cys repeats 1, 2, 3 and part of the TNFR-Cys repeat 4. Alternative38 amino acids at the C terminus. Missing part of TNFR-Cys repeat #4,the TM domain and the cytoplasmic domain. 5 MCD40Alt19 195 Contains 165N-terminal amino acids, including entire TNFR-Cys repeats 1, 2, 3 andpart of the TNFR-Cys repeat 4. Alternative 30 amino acids at the Cterminus. Missing part of TNFR-Cys repeat #4, the TM domain and thecytoplasmic domain. 6 MCD40Alt6Corecte 211 Contains 165 N-terminal aminoacids, including entire TNFR-Cys repeats 1, 2, 3 and part of theTNFR-Cys repeat 4. Insertion of 9 amino acids at the position #86 of thewild type protein (instead of the original Asn). Alternative 30 aminoacids at the C terminus. Missing part of TNFR-Cys repeat #4, the TMdomain and the cytoplasmic domain. SEQ ID NOS. 1 and 2 are from humansource origin. SEQ ID NOS: 4–6 are for mouse ortholog sequence.

“CD40R Variants products”—also referred at times as the “CD40R variantsproteins” or “CD40R variants polypeptides”—is an amino acid sequenceencoded by the CD40R variants nucleic acid sequences which is anaturally occurring mRNA sequence obtained as a result of alternativesplicing. The amino acid sequences may be a peptide, a protein, as wellas peptides or proteins having chemically modified amino acids (seebelow) such as a glycopeptide or glycoprotein. The CD40R variantsproducts are shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12. The termalso includes homologs (see below) of said sequences in which one ormore amino acids has been added, deleted, substituted (see below) orchemically modified (see below) as well as fragments (see below) of thissequence having at least 10 amino acids. As indicated above, thissequence refers to the extracellular domain of the CD40R.

“Fragments of CD40 variants nucleic acid sequences”—a partial sequenceof any one of SEQ ID NO: 1 to SEQ ID NO: 6 which includes the regionswhich contains the variation in nucleotides between the variant and theoriginal sequences. These regions (in the amino acid level) are asdepicted in the above Table 1.

“Fragments of CD40R variant product”—amino acid sequences coded by theabove nucleic acid fragment, containing regions by which the variantdiffers from the original sequence as indicated in Table 1.

“Nucleic acid sequence”‥a sequence composed of DNA nucleotides, RNAnucleotides or a combination of both types and may includes naturalnucleotides, chemically modified nucleotides and synthetic nucleotides.

“Amino acid sequence”‥a sequence composed of any one of the 20 naturallyappearing amino acids, amino acids which have been chemically modified(see below), or composed of synthetic amino acids.

“Homologues of variants/products”—amino acid sequences of variants inwhich one or more amino acids has been added, deleted or replaced. Thealtered amino acid shall be in regions where the variant differs fromthe original sequence, for example, according to the explanation inTable 1.

“Conservative substitution”—refers to the substitution of an amino acidin one class by an amino acid of the same class, where a class isdefined by common physicochemical amino acid side chain properties andhigh substitution frequencies in homologous proteins found in nature, asdetermined, for example, by a standard Dayhoff frequency exchange matrixor BLOSUM matrix. Six general classes of amino acid side chains havebeen categorized and include: Class 1 (Cys); Class II (Ser, Thr, Pro,Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys);Class V (ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example,substitution of an Asp for another class III residue such as Asn, Gln,or Glu, is a conservative substitution.

“Non-conservative substitution”—refers to the substitution of an aminoacid in one class with an amino acid from another class; for example,substitution of an Ala, a class II residue, with a class III residuesuch as Asp, Asn, Glu, or Gln.

“Chemically modified”—when referring to the product of the invention,means a product (protein) where at least one of its amino acid residesis modified either by natural processes, such as processing or otherpost-translational modifications, or by chemical modification techniqueswhich are well known in the art Among the numerous known modificationstypical, but not exclusive examples include:

-   -   acetylation, acylation, amidation, ADP-ribosylation,        glycosylation, GPI anchor formation, covalent attachment of a        lipid or lipid derivative, methylation, myristlyation,        pegylation, prenylation, phosphorylation, ubiqutination, or any        similar process.

“Biologically active”—refers to the variant product having some sort ofbiological activity, for example, capability of binding to the CD40ligand (CD40-L) or to other agonists of the original CD40R as known.

“Immunologically active” defines the capability of a natural,recombinant or synthetic varient product, or any fragment thereof, toinduce a specific immune response in appropriate animals or cells and tobind with specific antibodies. Thus, for example, an immunologicallyactive fragment of variant product denotes a fragment which retains someor all of the immunological properties of the variant product, e.g canbind specific anti-variant product antibodies or which can elicit animmune response which will generate such antibodies or causeproliferation of specific immune cells which produce variant.

“Optimal alignment”—is defined as an alignment giving the highestpercent identity score. Such alignment can be performed using a varietyof commercially available sequence analysis programs, such as the localalignment program LALIGN using a ktup of 1, default parameters and thedefault PAM. A preferred alignment is the one performed using theCLUSTAL-W program from MacVector™, operated with an open gap penalty of10.0, an extended gap penalty of 0.1, and a BLOSUM similarity matrix. Ifa gap needs to be inserted into a first sequence to optimally align itwith a second sequence, the percent identity is calculated using onlythe residues that are paired with a corresponding amino acid residue(i.e., the calculation does not consider residues in the secondsequences that are in the “gap” of the first sequence). In case ofalignments of known gene sequences with that of the new variant, theoptimal alignment invariably included aligning the identical parts ofboth sequences together, then keeping apart and unaligned the sectionsof the sequences that differ one from the other.

“Having at least 90% identity”—with respect to two amino acid or nucleicacid sequence sequences, refers to the percentage of residues that areidentical in the two sequences when the sequences are optimally aligned.Thus, 90% amino acid sequence identity means that 90% of the amino acidsin two or more optimally aligned polypeptide sequences are identical.

“Isolated nucleic acid molecule having an variant nucleic acidsequence”—is a nucleic acid molecule that includes the CD40R variantnucleic acid coding sequence. Said isolated nucleic acid molecule mayinclude the CD40R variant nucleic acid sequence as an independentinsert; may include the CD40R variant nucleic acid sequence fused to anadditional coding sequences, encoding together a fusion protein in whichthe variant coding sequence is the dominant coding sequence (forexample, the additional coding sequence may code for a signal peptide);the CD40R variant nucleic acid sequence may be in combination withnon-coding sequences, e.g., introns or control elements, such aspromoter and terminator elements or 5′ and/or 3′ untranslated regions,effective for expression of the coding sequence in a suitable host; ormay be a vector in which the CD40R variant protein coding sequence is aheterologous.

“Expression vector”—refers to vectors that have the ability toincorporate and express heterologous DNA fragments in a foreign cell.Many prokaryotic and eukaryotic expression vectors are known and/orcommercially available. Selection of appropriate expression vectors iswithin the knowledge of those having skill in the art.

“Deletion”—is a change in either nucleotide or amino acid sequence inwhich one or more nucleotides or amino acid residues, respectively, areabsent.

“Insertion” or “addition”—is that change in a nucleotide or amino acidsequence which has resulted in the addition of one or more nucleotidesor amino acid residues, respectively, as compared to the naturallyoccurring sequence.

“Substitution”—replacement of one or more nucleotides or amino acids bydifferent nucleotides or amino acids, respectively. As regards aminoacid sequences the substitution may be conservative or non-conservative.

“Antibody”—refers to IgG, IgM, IgD, IgA, and IgG antibody. Thedefinition includes polygonal antibodies or monoclonal antibodies. Thisterm refers to whole antibodies or fragments of the antibodiescomprising the antigen-binding domain of the anti-variant productantibodies, e.g. antibodies without the Fc portion, single chainantibodies, fragments consisting of essentially only the variable,antigen-binding domain of the antibody, etc.

“Treating a disease”—refers to administering a therapeutic substanceeffective to ameliorate symptoms associated with a disease, to lessenthe severity or cure the disease, or to prevent the disease fromoccurring.

“Detection”—refers to a method of detection of a disease, disorder,pathological or normal condition. This term may refer to detection of apredisposition to a disease as well as for establishing the prognosis ofthe patient by determining the severity of the disease.

“Probe”—the CD40R variant nucleic acid sequence, or a sequencecomplementary therewith, when used to detect presence of other similarsequences in a sample or of sequences having some homology with thissequence. The detection is carried out by identification ofhybridization complexes between the probe and the assayed sequence. Theprobe may be attached to a solid support or to a detectable label.

“Original CD40R”—the amino acid or nucleic acid sequence from which theCD40R variants of the invention have been varied as a result ofalternative slicing. The original nucleic sequence is the sequence ofthe human CD40 receptor depicted as CD40_(—)HUMAN Swiss Prot underAccession Number P25942 and the original amino acid sequence is thesequence encoded by it.

SUMMARY OF THE INVENTION

The present invention is based on the finding of six novel, naturallyoccurring splice variants of the CD40 receptor (CD40R), which arenaturally occurring sequences obtained by alternative splicing of theknown CD40R gene depicted as CD40_(—)HUMAN Swiss Prot under AccessionNumber P25942. The novel splice variants of the invention are not merelytruncated forms, or fragments of the known gene, but rather novelsequences which naturally occur within the body of individuals. Thesenovel variants in fact contain only the extracellular domains of theoriginal CD40R.

The term “alternative splicing” in the context of the present inventionand claims refers to: exon exclusion, and deletion of terminal sequencesin the variants as compared to the original sequence.

The novel CD40R variants of the invention retains the ligand-binding(extracellular) domain of the original CD40R, and thus are capable ofbinding to its ligands (for example the CD40-L) and decreasing in theindividual the amounts of such free ligands available for binding to theoriginal CD40R Thus the CD40R variants of the invention may act as a“scavengers” of the CD40 ligand, since they can bind those ligandswithout causing signal transduction due to said binding, and thuseffectively lowers the amount of said ligands. Since the variants aresecreted they can exert their scavenging effect even in body fluids.

The novel CD40R variants may also serve for detection purposes, i.e.their presence or level may be indicative of a disease, disorder,pathological or normal condition involving CD40 receptor such asinflammatory diseases, autoimmune diseases involving the immune system,pathological conditions, or alternatively the ratio between thevariants' level and the level of the original CD40R peptide from whichthey have been varied, or the ratio of any variants with respect to eachother may be indicative to such a disease, disorder, pathological ornormal condition.

For example, for detectional purposes, it is possible to establishdifferential expression of the CD40R variants in various tissues ascompared to the original CD40R. The variants (either each separately orboth) may be expressed mainly in one tissue, while the original CD40Rsequence from which they have been varied, may be expressed mainly inanother tissue. Understanding of the distribution of the variants ascompared to the original sequence or as compared to one another invarious tissues may be helpful in basic research, for understanding thephysiological function of the gene as well as may help in targetingpharmaceuticals or developing pharmaceuticals.

The detection may by determination of the presence or the level ofexpression of the CD40R variants within a specific cell population,comparing said presence or level between various cell types in a tissue,between different tissues and between individuals.

Thus the present invention provides by its first aspect, a novelisolated nucleic acid molecule comprising or consisting of the sequenceof any one of SEQ ID NO: 1 to SEQ ID NO: 6, fragments of said codingsequence having at least 20 nucleic acids or a molecule comprising asequence having at least 90%, identity to SEQ ID NO: 1 to SEQ ID NO: 6.

The present invention further provides a protein or polypeptidecomprising or consisting of an amino acid sequence encoded by any of theabove nucleic acid sequences, termed herein “CD40R variant product”, forexample, an amino acid sequence having the sequence as depicted in anyone of SEQ ID NO:7 to SEQ ID NO:12, fragments of the above amino acidsequence having a length of at least 10 amino acids coded by the abovefragments of the nucleic acid, as well as homologues of the above a=inoacid sequences in which one or more of the amino acid residues has beensubstituted (by conservative or non-conservative substitution) added,deleted, or chemically modified.

The present invention further provides nucleic acid molecule comprisingor consisting of a sequence which encodes the above amino acidsequences, (including the fragments and homologues of the amino acidsequences). Due to the degenerative nature of the genetic code, aplurality of alternative nucleic acid sequences, beyond the one depictedby any one of SEQ ID NO: 1 to SEQ ID NO:6, can code for the amino acidsequence of the invention. Those alternative nucleic acid sequence whichcode for the same amino acid sequences depicted any one of the sequencesSEQ ID NO:7 to SEQ ID NO: 12 are also an aspect of the of the presentinvention.

The present invention further provides expression vectors and cloningvectors comprising any of the above nucleic acid sequences, as well ashost cells transfected by said vectors.

The present invention still further provides pharmaceutical compositionscomprising, as an active ingredient, said nucleic acid molecules, saidexpression vectors, or said protein or polypeptide.

These pharmaceutical compositions are suitable for the treatment ofdiseases and pathological conditions, which can be ameliorated or curedby decreasing the levels of any one of the ligands of the originalCD40R. By the term “ligand” it is meant not only the CD40-L itself, butany other compounds such as TRAF3 TRAF2 which are known to interact withthe CD40 receptor. Examples of such diseases are: autoimmune diseasessuch as arthritis, rheumatoid arthritis, lupus, (SLE), and multiplesclerosis. The compositions may also be used to prevent development ofgraph versus host disease (GVHD) encountered after bone-marrowtansplantations.

The CD40R product may also be used for screening or constructingpharmaceuticals with improved specificity. Targeting pharmaceuticals tospecific tissues (which express one variant), or targeting them againstone condition (in which a particular variant is expressed) may be aidedby the variants of the invention which enable screening or constructingpharmaceuticals with improved tissue, or condition specificity.

By a second aspect, the present invention provides a nucleic acidmolecule comprising or consisting of a non-coding sequence which iscomplementary to that of any one of SEQ ID NO:1 to SEQ ID NO:6, orcomplementary to a sequence having at least 90% identity to saidsequences or a fragment of said sequences. The complementary sequencemay be a DNA sequence which hybridizes with the sequences of SEQ of IDNO: 1 to SEQ ID NO:6 or hybridizes to a portion of those sequenceshaving a length sufficient to inhibit the transcription of thecomplementary sequences. The complementary sequence may be a DNAsequence which can be transcribed into an mRNA being an antisense to themRNA transcribed from SEQ ID NO:1 to SEQ ID NO:6 or into an mRNA whichis an antisense to a fragment of the mRNA transcribed from SEQ ID NO:1to SEQ ID NO:6 which has a length sufficient to hybridize with the mRNAtranscribed from any one of SEQ ID NO:1 to SEQ ID NO:6, so as to inhibitits translation. The complementary sequence may also be the mRNA or thefragment of the mRNA itself.

The nucleic acids of the second aspect of the invention may be used fortherapeutic or diagnostic applications for example as probes used forthe detection of the CD40R variants of the invention. The presence ofthe CD40R variants transcripts or the level of the variants transcriptsmay be indicative of a multitude of diseases, disorders and variouspathological as well as normal conditions. In addition, the ratio of thelevel of the transcripts of the variants of the invention may also becompared to that of the transcripts of the original CD40R sequences fromwhich they have been varied or the ratio of each other, and said ratiomay be indicative to a multitude of diseases, disorders and variouspathological and normal conditions. Comparison may be aided by the factthat while the known CD40 receptor has both cytoplasmic andextracellular regions the variants of the invention have essentiallyonly extracellular regions and are thus secreted soluble.

The present invention also provides expression vectors comprising anyone of the above defined complementary nucleic acid sequences and hostcells transfected with said nucleic acid sequences or vectors, beingcomplementary to those specified in the first aspect of the invention.

The invention also provides anti-variant product antibodies, namelyantibodies directed against the CD40R variants products whichspecifically bind to said CD40R variants products. Said antibodies areuseful both for diagnostic and therapeutic purposes. For example saidantibodies may be as an active ingredient in a pharmaceuticalcomposition as will be explained below.

The present invention also provides pharmaceutical compositionscomprising, as an active ingredient, the nucleic acid molecules whichcomprise or consist of said complementary sequences, or of a vectorcomprising said complementary sequences. The pharmaceutical compositionthus provides pharmaceutical compositions comprising, as an activeingredient, said anti-variant product antibodies.

According to the third aspect of the invention the present inventionprovides methods for detecting the level of the transcripts (mRNA) ofsaid CD40R variants product in a body fluid sample, or in a specifictissue sample, for example by use of probes comprising or consisting ofsaid coding sequences; as well as methods for detecting levels ofexpression of said product in tissue, e.g. by the use of antibodiescapable of specifically reacting with the CD40 variants products of theinvention. Detection of the level of the expression of the CD40 variantsof the invention in particular as compared to that of the originalsequence from which it was varied or compared to each other may beindicative of a plurality of physiological or pathological conditions.

The method, according to this latter aspect, for detection of a nucleicacid sequence which encodes the CD40R variants products in a biologicalsample, comprises the steps of:

-   -   (a) providing a probe comprising at least one of the nucleic        acid sequences defined above;    -   (b) contacting the biological sample with said probe under        conditions allowing hybridization of nucleic acid sequences        thereby enabling formation of hybridization complexes;    -   (c) detecting hybridization complexes, wherein the presence of        the complex indicates the presence of nucleic acid sequence        encoding the CD40R variants products in the biological sample.

The method as described above is qualitative, i.e. indicates whether thetranscripts are present in or absent from the sample. The method canalso be quantitative, by determining the level of hybridizationcomplexes and then calibrating said levels to determining levels oftranscripts of the desired variants in the sample.

Both qualitative and quantitative determination methods can be used fordiagnostic, prognostic and therapy planning purposes.

By a preferred embodiment the probe is part of a nucleic acid chip usedfor detection purposes, i.e. the probe is a part of an array of probeseach present in a known location on a solid support.

The nucleic acid sequences used in the above method may be a DNAsequence an RNA sequence, etc; they may be a coding or a sequence or asequence complementary thereto (for respective detection of RNAtranscripts or coding-DNA sequences). By quantization of the level ofhybridization complexes and calibrating the quantified results it ispossible also to detect the level of the transcripts in the sample.

Methods for detecting mutations in the region coding for the CD40Rvariants products are also provided, which may be methods carried-out ina binary fashion, namely merely detecting whether there is anymismatches between the normal variant nucleic acid sequence of theinvention and the one present in the sample, or carried-out byspecifically detecting the nature and location of the mutation.

The present invention also concerns a method for detecting the CD40Rvariants products in a biological sample, comprising the steps of:

-   -   (a) contacting with said biological sample the antibody of the        invention, thereby forming an antibody-antigen complex; and    -   (b) detecting said antibody-antigen complex    -   wherein the presence of said antibody-antigen complex correlates        with the presence of the CD40R variants products in said        biological sample.

As indicated above, the method can be quantitized to determine the levelor the amount of the CD40R variants in the sample, alone or incomparison to the level of the original CD40R amino acid sequence fromwhich it was varied, and qualitative and quantitative results may beused for diagnostic, prognostic and therapy planning purposes.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows multiple alignment of four amino acid sequences ID NOS: 3–6of mouse origin (depicted in SEQ ID NO:9 to SEQ ID NO:12 to each otherand to the original sequence;

FIG. 2 shows Western blot analysis (top) of St-9 cells infected withCD40 expression vectors and an abnormal spectrum at 280 nm as a functionof time;

FIG. 3 shows Western blot analysis of CD40 secreted from St-0 cellstransfected with an expression vector;

FIG. 4 shows comparison by alignment of the original CD40R nucleic acidsequence as obtained from Swiss Prot Accession No. 25942 and SEQ IDNO:1;

FIG. 5 shows a comparison of the original CD40 nucleic acid sequence andSEQ ID NO:2; and

FIG. 6 shows comparison by alignment of the original CD40R amino acidsequence and SEQ ID NO:7.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE I CD40R VariantsNucleic Acid Sequence

The nucleic acid sequences of the invention include nucleic acidsequences which encode CD40R variants products and fragments and analogsthereof. The nucleic acid sequences may alternatively be sequencescomplementary to the above coding sequences, or to regions of saidcoding sequence. The length of the complementary sequences is sufficientto avoid the expression of the coding sequence. The nucleic acidsequences may be in the form of RNA or in the form of DNA, and includemessenger RNA, synthetic RNA and DNA, cDNA and genomic DNA. The DNA maybe double-stranded or simple-stranded, and if single-stranded may be thecoding strand or the non-coding (anti-sense, complementary) strand. Thenucleic acid sequences may also both include dNTPs, rNTPs as well as nonnaturally occurring sequences. The sequence may also be a part of ahybrid between an amino acid sequence and a nucleic acid sequence.

In a general embodiment, the nucleic acid sequence has at least 90%,identity with any one of the sequence identified as SEQ ID NO:1 to SEQID NO:6.

The nucleic acid sequences may include the coding sequence by itself. Byanother alternative the coding region may be in combination withadditional coding sequences, such as those coding for fusion protein orsignal peptides, in combination with non-coding sequences, such asintrons and control elements, promoter and terminator elements or 5′and/or 3′ untranslated regions, effective for expression of the codingsequence in a suitable host, and/or in a vector or host environment inwhich the variant nucleic acid sequences is introduced as a heterologoussequence.

The nucleic acid sequences of the present invention may also have theCD40R variants products coding sequences fused in-frame to a markersequence which allows for purification of the variant product. Themarker sequence may be, for example, a hexahistidine tag to provide forpurification of the mature polypeptide fused to the marker in the caseof a bacterial host, or, the marker sequence may be a hemagglutinin (HA)tag when a mammalian host, e.g. COS-7 cells, is used. The HA tagcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson, I., et al. Cell 37:767 (1984)).

Also included in the scope of the invention are fragments as definedabove also referred to herein as oligonucleotides, typically having atleast 20 bases, preferably 20–30 bases corresponding to a region of thecoding-sequence nucleic acid sequence. The fragments may be used asprobes, primers, and when complementary also as antisense agents, andthe like, according to known methods.

As indicated above, the nucleic acid sequence may be substantially adepicted in SEQ ID NO:1 to SEQ ID NO:6 or fragments thereof or sequenceshaving at least 90% identity to the above sequence as explained above.Alternatively, due to the degenerative nature of the genetic code, thesequence may be a sequence coding for any one of the amino acid sequenceof SEQ ID NO:7 or SEQ ID NO:12, or fragments or analogs of said aminoacid sequence.

A. Preparation of Nucleic Acid Sequences

The nucleic acid sequences may be obtained by screening cDNA librariesusing oligonucleotide probes which can hybridize to or PCR-amplifynucleic acid sequences which encode the CD40R variants productsdisclosed above. cDNA libraries prepared from a variety of tissues arecommercially available and procedures for screening and isolating cDNAclones are well-known to those of skill in the art. Such techniques aredescribed in, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2nd Edition), Cold Spring Harbor Press, Plainview,N.Y. and Ausubel FM et al. (1989) Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y.

The nucleic acid sequences may be extended to obtain upstream anddownstream sequences such as promoters, regulatory elements, and 5′ and3′ untranslated regions (UTRs). Extension of the available transcriptsequence may be performed by numerous methods known to those of skill inthe art, such as PCR or primer extension (Sambrook et al., supra), or bythe RACE method using, for example, the Marathon RACE kit (Clontech,Cat. # K1802-1).

Alternatively, the technique of “restriction-site” PCR (Gobinda et al.PCR Methods Applic. 2:318–22, (1993)), which uses universal primers toretrieve flanking sequence adjacent a known locus, may be employed.First, genomic DNA is amplified in the presence of primer to a linkersequence and a primer specific to the known region. The amplifiedsequences are subjected to a second round of PCR with the same linkerprimer and another specific primer internal to the first one. Productsof each round of PCR are transcribed with an appropriate RNA polymeraseand sequenced using reverse transcriptase.

Inverse PCR can be used to amplify or extend sequences using divergentprimers based on a known region (Triglia, T. et al., Nucleic Acids Res.16:8186, (1988)). The primers may be designed using OLIGO(R) 4.06 PrimerAnalysis Software (1992; National Biosciences Inc, Plymouth, Minn.), oranother appropriate program, to be 22–30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68–72° C. The method uses several restriction enzymesto generate a suitable fragment in the known region of a gene. Thefragment is then circularized by intramolecular ligation and used as aPCR template.

Capture PCR (Lagerstrom, M. et al., PCR Methods Applic. 1:111–19,(1991)) is a method for PCR amplification of DNA fragments adjacent to aknown sequence in human and yeast artificial chromosome DNA. Capture PCRalso requires multiple restriction enzyme digestions and ligations toplace an engineered double-stranded sequence into a flanking part of theDNA molecule before PCR.

Another method which may be used to retrieve flanking sequences is thatof Parker, J. D., et al., Nucleic Acids Res., 19:3055-60, (1991)).Additionally, one can use PCR, nested primers and PromoterFinder™libraries to “walk in” genomic DNA (PromoterFinder™; Clontech, PaloAlto, Calif.). This process avoids the need to screen libraries and isuseful in finding intron/exon junctions. Preferred libraries forscreening for full length cDNAs are ones that have been size-selected toinclude larger cDNAs. Also, random primed libraries are preferred inthat they will contain more sequences which contain the 5′ and upstreamregions of genes.

A randomly primed library may be particularly useful if an oligo d(T)library does not yield a full-length cDNA. Genomic libraries are usefulfor extension into the 5′ nontranslated regulatory region.

The nucleic acid sequences and oligonucleotides of the invention canalso be prepared by solid-phase methods, according to known syntheticmethods. Typically, fragments of up to about 100 bases are individuallysynthesized, then joined to form continuous sequences up to severalhundred bases.

B. Use of CD40R Variants Nucleic Acid Sequences for the Production ofCD40R Variants Products

In accordance with the present invention, nucleic acid sequencesspecified above may be used as recombinant DNA molecules that direct theexpression of CD40R variant products.

As will be understood by those of skill in the art, it may beadvantageous to produce CD40R variants product-encoding nucleotidesequences possessing codons other than those which appear in SEQ ID NO:1to SEQ ID NO:6 which are those which naturally occur in the humangenome. Codons preferred by a particular prokaryotic or eukaryotic host(Murray, E. et al. Nuc Acids Res., 17:477-508, (1989)) can be selected,for example, to increase the rate of variant product expression or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, than transcripts produced from naturally occurringsequence.

The nucleic acid sequences of the present invention can be engineered inorder to alter a CD40R variants products coding sequences for a varietyof reasons, including but not limited to, alterations which modify thecloning, processing and/or expression of the product. For example,alterations may be introduced using techniques which are well known inthe art, e.g., site-directed mutagenesis, to insert new restrictionsites, to alter glycosylation patterns, to change codon preference, etc.

The present invention also includes recombinant constructs comprisingone or more of the sequences as broadly described above. The constructscomprise a vector, such as a plasmid or viral vector, into which nucleicacid sequences of the invention have been inserted, in a forward orreverse orientation. In a preferred aspect of this embodiment, theconstructs further comprise regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and are commercially available. Appropriate cloning and expressionvectors for use with prokaryotic and eukaryotic hosts are also describedin Sambrook et al., (supra).

The present invention also relates to host cells which are geneticallyengineered with vectors of the invention, and the production of theproduct of the invention by recombinant techniques. Host cells aregenetically engineered (i.e., transduced, transformed or transfected)with the vectors of this invention which may be, for example, a cloningvector or an expression vector. The vector may be, for example, in theform of a plasmid, a viral particle, a phage, etc. The engineered hostcells can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying the expression of the variant nucleic acid sequence. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to those skilled in the art.

The nucleic acid sequences of the present invention may be included inany one of a variety of expression vectors for expressing a product.Such vectors include chromosomal, nonchromosomal and synthetic DNAsequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;baculovirus; yeast plasmids; vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. However, any other vector may be used as longas it is replicable and viable in the host. The appropriate DNA sequencemay be inserted into the vector by a variety of procedures. In general,the DNA sequence is inserted into an appropriate restrictionendonuclease site(s) by procedures known in the art. Such procedures andrelated sub-cloning procedures are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate transcription control sequence (promoter) to direct mRNAsynthesis. Examples of such promoters include: LTR or SV40 promoter, theE. coli lac or trp promoter, the phage lambda PL promoter, and otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses. The expression vectors also containsa ribosome binding site for translation initiation, and a transcriptionterminator. The vector may also include appropriate sequences foramplifying expression. In addition, the expression vectors preferablycontain one or more selectable marker genes to provide a phenotypictrait for selection of transformed host cells such as dihydrofolatereductase or neomycin resistance for eukaryotic cell culture, or such astetracycline or ampicillin resistance in E. coli.

The vectors containing the appropriate DNA sequence as described above,as well as an appropriate promoter or control sequence, may be employedto transform an appropriate host to permit the host to express theprotein. Examples of appropriate expression hosts include: bacterialcells, such as E. coli, Streptomyces, Salmonella typhimurium; fungalcells, such as yeast; insect cells such as Drosophila and SpodopteraSf9; animal cells such as CHO, COS, HEK 293 or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein. The invention is not limited by the host cellsemployed.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the CD40R variant product. Forexample, when large quantities of CD40R variant product are needed forthe induction of antibodies, vectors which direct high level expressionof fusion proteins that are readily purified may be desirable. Suchvectors include, but are not limited to, multifunctional E. coli cloningand expression vectors such as Bluescript(R) (Stratagene), in which theCD40R variants polypeptides coding sequence may be ligated into thevector in-frame with sequences for the amino-terminal Met and thesubsequent 7 residues of beta-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke & Schuster J. Biol. Chem.264:5503-5509, (1989)); pET vectors (Novagen, Madison Wis.); and thelike.

In the yeast Saccharomyces cerevisiae a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase and PGH may be used. For reviews, see Ausubel et al. (supra) andGrant et al., (Methods in Enzymology 153:516-544, (1987)).

In cases where plant expression vectors are used, the expression of asequence encoding variant products may be driven by any of a number ofpromoters. For example, viral promoters such as the ³⁵S and 19Spromoters of CaMV (Brisson et al., Nature 310:511-514. (1984)) may beused alone or in combination with the omega leader sequence from TMV(Takamatsu et al., EMBO J., 6:307-311, (1987)). Alternatively, plantpromoters such as the small subunit of RUBISCO (Coruzzi et al., EMBO J.3:1671-1680, (1984); Broglie et al., Science 224:838-843, (1984)); orheat shock promoters (Winter J and Sinibaldi R. M., Results Probl. CellDiffer., 17:85-105, (1991)) may be used. These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. For reviews of such techniques, seeHobbs S. or Murry L. E. (1992) in McGraw Hill Yearbook of Science andTechnology, McGraw Hill, New York, N.Y., pp 191-196; or Weissbach andWeissbach (1988) Methods for Plant Molecular Biology, Academic Press,New York, N.Y., pp 421-463.

CD40R variants products may also be expressed in an insect system. Inone such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The CD40R variants productscoding sequence may be cloned into a nonessential region of the virus,such as the polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of CD40R variants coding sequences willrender the polyhedrin gene inactive and produce recombinant viruslacking coat protein coat. The recombinant viruses are then used toinfect S. frugiperda cells or Trichoplusia larvae in which variantprotein is expressed (Smith et al., J. Virol. 46:584, (1983); Engelhard,E. K. et al., Proc. Nat. Acad. Sci. 91:3224-7, (1994)).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, 5 CD40R variants products coding sequences may be ligated intoan adenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a nonessential E1or E3 region of the viral genome will result in a viable virus capableof expressing variant protein in infected host cells (Logan and Shenk,Proc. Natl. Acad. Sci. 81:3655-59, (1984). In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells.

Specific initiation signals may also be required for efficienttranslation of variants products coding sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where CD40Rvariants products coding sequence, its initiation codon and upstreamsequences are inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only coding sequence, or a portion thereof, is inserted,exogenous transcriptional control signals including the ATG initiationcodon must be provided. Furthermore, the initiation codon must be in thecorrect reading frame to ensure transcription of the entire insert.Exogenous transcriptional elements and initiation codons can be ofvarious origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of enhancers appropriate tothe cell system in use (Scharf, D. et al., (1994) Results Probl. CellDiffer., 20:125-62, (1994); Bittner et al., Methods in Enzymol153:516-544, (1987)).

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., and Battey, 1.(1986) Basic Methods in Molecular Biology). Cell-free translationsystems can also be employed to produce polypeptides using RNAs derivedfrom the DNA constructs of the present invention.

A host cell strain may be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Post-translational processingwhich cleaves a “pre-pro” form of the protein may also be important forcorrect insertion, folding and/or function. Different host cells such asCHO, HeLa, MDCK, 293, WI38, etc. have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and maybe chosen to ensure the correct modification and processing of theintroduced, foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressvariant products may be transformed using expression vectors whichcontain viral origins of replication or endogenous expression elementsand a selectable marker gene. Following the introduction of the vector,cells may be allowed to grow for 1-2 days in an enriched media beforethey are switched to selective media. The purpose of the selectablemarker is to confer resistance to selection, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clumps of stably transformed cells can beproliferated using tissue culture techniques appropriate to the celltype.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler M., et al., Cell 11:223-32, (1977)) and adeninephosphoribosyltransferase (Lowy I., et al., Cell 22:817-23, (1980))genes which can be employed in tk- or aprt-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler M., et al., Proc. Natl. Acaci. Sci. 77:3567-70,(1980)); npt, which confers resistance to the aminoglycosides neomycinand G418 (Colbere-Garapin, F. et al., J. Mol. Biol., 150:1-14, (1981))and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman S.C. and R. C. Mulligan, Proc. Natl. Acad. Sci. 85:8047-51, (1988)). Theuse of visible markers has gained popularity with such markers asanthocyanins, beta-glucuronidase and its substrate, GUS, and luciferaseand its substrates, luciferin and ATP, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et. al., Methods Mol. Biol., 55:121-131, (1995)).

Host cells transformed with nucleotide sequences encoding CD40R variantsproducts may be cultured under conditions suitable for the expressionand recovery of the encoded protein from cell culture. The productproduced by a recombinant cell may be secreted or containedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining nucleic acid sequences encoding CD40R variants products canbe designed with signal sequences which direct secretion of CD40Rvariants products through a prokaryotic or eukaryotic cell membrane.

The CD40R variants products may also be expressed as recombinantproteins with one or more additional polypeptide domains added tofacilitate protein purification. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle, Wash.). The inclusion of aprotease-cleavable polypeptide linker sequence between the purificationdomain and CD40R variants products is useful to facilitate purification.One such expression vector provides for expression of a fusion proteincompromising a variant polypeptide fused to a polyhistidine regionseparated by an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography, as described in Porath, et al., Protein Expression andPurification, 3:263-281, (1992)) while the enterokinase cleavage siteprovides a means for isolating variant polypeptide from the fusionprotein. pGEX vectors (Promega, Madison, Wis.) may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption to ligand-agarosebeads (e.g., glutathione-agarose in the case of GST-fusions) followed byelution in the presence of free ligand.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, orother methods, which are well know to those skilled in the art.

The CD40R variants products can be recovered and purified fromrecombinant cell cultures by any of a number of methods well known inthe art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography, and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

C. Use of Varient to Produce Proteins

C.1 Separation:

Sf-9 cells were infected with sCD40 (soluble CD40) expressingbaculovirus (Ac-sCD40) comprising the nucleic acid sequence of SEQ IDNO:3 at MOI of 2. The cells were grown in 28° C. at Continuous shaking(90 rpm). At 60 hours post infection (hpi) the medium was collected andcells were separated from the medium by centrifugation at 5000 RPM for 5minutes. 10 ml medium was separated using cation exchange chromatographywith SP-Sepharose column. The Column was equilibrated with PBS pH-6.5and following loading of the sample on the column the column was washedwith PBS to elute the unbound proteins (flow through fraction). Elutionwas done with increasing concentration of NaCl at flow rate of 2 ml/min(5% NaCl/min).

The different fractions were subjected to SDS-PAGE electrophoresis andto western blotting using anti mCD40 antibody, and the results are shownin FIG. 2.

C. 1 Secretion:

Sf-9 cells were infected with sCD40 expressing baculovirus (Ac-sCD40) atMOI of 2. The cells were grown at 28° C. at continuous shaking (90 rpm)and 1 ml samples were collected at 24, 48 and 60 hours post infection(hpi). Following centrifugation Cells pellet was lysed with lysis buffer(50 mM Tris pH 7.5. 1% triton X100, and protease inhibitor cocktail) at4° C. for 30 min and sonicated for 30 seconds. The sample wascentrifuged for 10 minutes at 14000 rmp and the sup was designatedPellet. 40 μl of the pellet preparation and of the medium (DesignatedMedium) were supplemented with sample buffer and electrophoreses on a15° SDS-PAGE. Following electrophoresis the gel was subjected to a semidry protein transfer onto a nitrocellulose membrane. The protein.Finally, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

C. Use of Varient to Produce Proteins

C.1 Separation:

Sf-9 cells were infected with sCD40 (soluble CD40) expressingbaculovirus (Ac-sCD40) comprising the nucleic acid sequence of SEQ IDNO:3 at MOI of 2. The cells were grown in 28° C. at continuous shaking(90 rpm). At 60 hours post infection (hpi) the medium was collected andcells were separated from the medium by centrifugation at 5000 RPM for 5minutes. 10 ml medium was separated using cation exchange chromatographywith SP-Sepharose column. The Column was equilibrated with PBS pH-6.5and following loading of the sample on the column the column was washedwith PBS to elute the unbound proteins (flow through fraction). Elutionwas done with increasing concentration of NaCl at flow rate of 2 ml/min(5% NaCl/min).

The different fractions were subjected to SDS-PAGE electrophoresis andto western blotting using anti mCD40 antibody, and the results are shownin FIG. 2.

C.1 Secretion:

Sf-9 cells were infected with sCD40 expressing baculovirus (Ac-sCD40) atMOI of 2. The cells were grown at 28° C. at continuous shaking (90 rpm)and 1 ml samples were collected at 24, 48 and 60 hours post infection(hpi). Following centrifugation Cells pellet was lysed with lysis buffer(50 mM Tris pH 7.5, 1% triton X100, and protease inhibitor cocktail) at4° C. for 30 min and sonicated for 30 seconds. The sample wascentrifuged for 10 minutes at 14000 rmp and the sup was designatedPellet. 40 μl of the pellet preparation and of the medium (DesignatedMedium) were supplemented with sample buffer and electrophoreses on a15% SDS-PAGE. Following electrophoresis the gel was subjected to a semidry protein transfer onto a nitrocellulose membrane. The membrane wasincubated with anti mCD40 antibody for 2 hours and with secondary antirabbit antibody for an additional 1 hour.

Detection of the signal was done using a commercial western blotdetection kit, and the results are shown in FIG. 3.

D. Diagnostic Applications Utilizing Nucleic Acid Sequences

The nucleic acid sequences of the present invention may be used for avariety of diagnostic purposes. The nucleic acid sequences may be usedto detect and quantitate expression of the CD40R variant in patient'scells, e.g. biopsied lo tissues, by detecting the presence of mRNAcoding for the CD40R variants products. Alternatively, the assay may beused to detect the soluble variants in the serum or blood. This assaytypically involves obtaining total mRNA from the tissue or serum andcontacting the mRNA with a nucleic acid probe. The probe is a nucleicacid molecule of at least 20 nucleotides, preferably 20-30 nucleotides,capable of specifically hybridizing with a sequence included within thesequence of a nucleic acid molecule encoding the CD40R variant productunder hybridizing conditions, detecting the presence of mRNA hybridizedto the probe, and thereby detecting the expression of variant. Thisassay can be used to distinguish between absence, presence, and excessexpression of CD40R variants products and to monitor levels of CD40Rvariants expression during therapeutic intervention. In addition, theassay may be used to compare the levels of the CD40R variant of theinvention to the levels of the original CD40R sequence from which it hasbeen varied or to levels of each other, which comparison may have somephysiological meaning.

The invention also contemplates the use of the nucleic acid sequences asa diagnostic for diseases resulting from inherited defective variantssequences, or diseases in which the ratio of the amount of the originalCD40R sequence from which the CD40R variants were varied to the novelCD40R variants of the invention is altered. These sequences can bedetected by comparing the sequences of the defective (i.e., mutant)CD40R variants coding region with that of a normal coding region.Association of the sequence coding for mutant CD40R variants productswith abnormal variants products activity may be verified. In addition,sequences encoding mutant CD40R variants products can be inserted into asuitable vector for expression in a functional assay system (e.g.,colorimetric assay, complementation experiments in a variant proteindeficient strain of HEK2993 cells) as yet another means to verify oridentify mutations. Once mutant genes have been identified, one can thenscreen populations of interest for carriers of the mutant gene.

Individuals carrying mutations in the nucleic acid sequences of thepresent invention may be detected at the DNA level by a variety oftechniques. Nucleic acids used for diagnosis may be obtained from apatients cells, including but not limited to such as from blood, urine,saliva, placenta, tissue biopsy and autopsy material. Genomic DNA may beused directly for detection or may be amplified enzymatically by usingPCR (Saiki, et al., Nature 324:163-166, (1986)) prior to analysis. RNAor cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid of the present invention canbe used to identify and analyze mutations in the gene of the presentinvention. Deletions and insertions can be detected by a change in sizeof the amplified product in comparison to the normal genotype.

Point mutations can be identified by hybridizing amplified DNA toradiolabeled RNA of the invention or alternatively, radiolabeledantisense DNA sequences of the invention. Sequence changes at specificlocations may also be revealed by nuclease protection assays, such RNaseand S1 protection or the chemical cleavage method (e.g. Cotton, el alProc. Natl Acad. Sci. USA, 85:4397-4401, (1985)), or by differences inmelting temperatures. “Molecular beacons” (Kostrikis L. G. et al.,Science 279:1228-1229, (1998)), hairpin-shaped, single-strandedsynthetic oligo-nucleotides containing probe sequences which arecomplementary to the nucleic acid of the present invention, may also beused to detect point mutations or other sequence changes as well asmonitor expression levels of variant product. Such diagnostics would beparticularly useful for prenatal testing.

Another method for detecting mutations uses two DNA probes which aredesigned to hybridize to adjacent regions of a target, with abuttingbases, where the region of known or suspected mutation(s) is at or nearthe abutting bases. The two probes may be joined at the abutting bases,e.g., in the presence of a ligase enzyme, but only if both probes arecorrectly base paired in the region of probe junction. The presence orabsence of mutations is then detectable by the presence or absence ofligated probe.

Also suitable for detecting mutations in the CD40R variants productscoding sequences are oligonucleotide array methods based on sequencingby hybridization (SBH), as described, for example, in U.S. Pat. No.5,547,839. In a typical method, the DNA target analyte is hybridizedwith an array of oligonucleotides formed on a microchip. The sequence ofthe target can then be “read” from the pattern of target binding to thearray.

E. Therapeutic Applications of Nucleic Acid Sequences

Nucleic acid sequences of the invention may also be used for therapeuticpurposes. Turning first to the second aspect of the invention (i.e.inhibition of expression of CD40R variants), expression of CD40Rvariants products may be modulated through antisense technology, whichcontrols gene expression through hybridization of complementary nucleicacid sequences, i.e. antisense DNA or RNA, to the control, 5′ orregulatory regions of the gene encoding variant product. For example,the 5′ coding portion of the nucleic acid sequence sequence which codesfor the product of the present invention is used to design an antisenseoligonucleotide of from about 10 to 40 base pairs in length.Oligonucleotides derived from the transcription start site, e.g. betweenpositions −10 and +10 from the start site, are preferred. An antisenseDNA oligonucleotide is designed to be complementary to a region of thenucleic acid sequence involved in transcription (Lee et al., Nucl.Acids, Res., 6:3073, (1979); Cooney et al. Science 241:456, (1988); andDervan et al., Science 251:1360, (1991)), thereby preventingtranscription and the production of the variant products. An antisenseRNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the variant products (Okano J.Neurochem. 56:560, (1991)). The antisense constructs can be delivered tocells by procedures known in the art such that the antisense RNA or DNAmay be expressed in vivo. The antisense may be antisense mRNA or DNAsequence capable of coding such antisense mRNA. The antisense mRNA orthe DNA coding thereof can be complementary to the full sequence ofnucleic acid sequences coding for the CD40R variant protein or to afragment of such a sequence which is sufficient to inhibit production ofa protein product. Antisense technologies can also be used forinhibiting expression of one variant as compared to the other, orinhibiting the expression of the variant/s as compared to the originalsequence.

Turning now to the first aspect of the invention, i.e. expression ofCD40R variants, expression of CD40R variants products may be increasedby providing coding sequences for coding for said CD40R variantsproducts under the control of suitable control elements ending itsexpression in the desired host.

The nucleic acid sequences of the invention may be employed incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the compound, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The products of the invention may also be employed in accordance withthe present invention by expression of such polypeptides in vivo, whichis often referred to as “gene therapy.” Cells from a patient may beengineered with a nucleic acid sequence (DNA or RNA) encoding apolypeptide ex vivo, with the engineered cells then being provided to apatient to be treated with the polypeptide. Such methods are well-knownin the art. For example, cells may be engineered by procedures known inthe art by use of a retroviral particle containing RNA encoding apolypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by procedures known in the art. As known in the art,a producer cell for producing a retroviral particle containing RNAencoding the polypeptides of the present invention may be administeredto a patient for engineering cells in vivo and expression of thepolypeptide in vivo. These and other methods for administering productsof the present invention by such method should be apparent to thoseskilled in the art from the teachings of the present invention. Forexample, the expression vehicle for engineering cells may be other thana retrovirus, for example, an adenovirus which may be used to engineercells in vivo after combination with a suitable delivery vehicle.

Retroviruses from which the retroviral plasmid vectors mentioned abovemay be derived include, but are not limited to, Moloney Murine LeukemiaVirus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus,human immunodeficiency virus, adenovirus, Myeloproliferative SarcomaVirus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, psi-2,psi-AM, PA12, T19-14X, VT-19-17-H2, psi-CRE, psi-CRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller (Human GeneTherapy, Vol. 1, pg. 5-14, (1990)). The vector may transduce thepackaging cells through any means known in the art. Such means include,but are not limited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

The genes introduced into cells may be placed under the control ofinducible promoters, such as the radiation-inducible Egr-1 promoter,(Maceri, H. J., et al., Cancer Res., 56(19):4311 (1996)), to stimulatevariant production or antisense inhibition in response to radiation,eg., radiation therapy for treating tumors.

EXAMPLE II CD40R Variants Products

The substantially purified CD40R variant product of the invention hasbeen defined above as the product coded from the nucleic acid sequenceof the invention. Preferably the amino acid sequence is an amino acidsequence having at least 90% identity the sequence identified as SEQ IDNO:7 to SEQ ID NO:12. The protein or polypeptide may be in mature and/ormodified form, also as defined above, for example, modified by cleavageof the leader sequence. Also contemplated are protein fragments havingat least 10 contiguous amino acid residues, preferably at least 10-20residues, derived from the CD40R variant products, as well as homologuesas explained above.

The sequence variations are preferably those that are consideredconserved substitutions, as defined above. Thus, for example, a proteinwith a sequence having at least 90% sequence identity with the productsidentified as SEQ ID NO:7 to SEQ ID NO:12, preferably by utilizingconserved substitutions as defined above is also part of the invention,and provided that it is not identical to the original peptide from whichit has been varied (typically the substitutions are in regions where thevariant differs from the original sequence as for example in Table 1).In a more specific embodiment, the protein has or contains the sequenceidentified SEQ ID NO:7 to SEQ ID NO:12. The CD40R variants products maybe (i) one in which one or more of the amino acid residues in a sequencelisted above are substituted with a conserved or non-conserved aminoacid residue (preferably a conserved amino acid residue), or (ii) one inwhich one or more of the amino acid residues includes a substituentgroup, or (iii) one in which the CD40R variants products is fused withanother compound, such as a compound to increase the half-life of theprotein (for example, polyethylene glycol (PEG)), or a moiety whichserves as targeting means to direct the protein to its target tissue ortarget cell population (such as an antibody), or (iv) one in whichadditional amino acids are fused to the CD40R variant product. Suchfragments, variants and derivatives are deemed to be within the scope ofthose skilled in the art from the teachings herein.

A. Preparation of CD40R Variants Products

Recombinant methods for producing and isolating the CD40R variantproducts, and fragments of the protein are described above.

In addition to recombinant production, fragments and portions of variantproducts may be produced by direct peptide synthesis using solid-phasetechniques (cf. Stewart et al., (1969) Solid-Phase Peptide Synthesis, WHFreeman Co, San Francisco; Merrifield J., J. Am. Chem. Soc.,85:2149-2154, (1963)). In vitro peptide synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City, Calif.) in accordance with the instructions providedby the manufacturer. Fragments of CD40R variants products may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

B. Therapeutic uses and Compositions Utilizing the CD40R VariantsProducts

The CD40R variants products of the invention are generally useful intreating diseases and disorders which can be cured or ameliorated bylowering the level of any of the CD40 ligands. Examples of such diseasesare various autoimmune diseases as well as GVHD.

CD40R variant products or fragments may be administered by any of anumber of routes and methods designed to provide a consistent andpredictable concentration of compound at the target organ or tissue. Theproduct-containing compositions may be administered alone or incombination with other agents, such as stabilizing compounds, and/or incombination with other pharmaceutical agents such as drugs or hormones.

CD40R variants product-containing compositions may be administered by anumber of routes including, but not limited to oral, intravenous,intramuscular, transdermal, subcutaneous, topical, sublingual, or rectalmeans as well as by nasal application. CD40R variant product-containingcompositions may also be administered via liposomes. Such administrationroutes and appropriate formulations are generally known to those ofskill in the art.

The CD40R variants products can be given via intravenous orintraperitoneal injection. Similarly, the product may be injected toother localized regions of the body. The product may also beadministered via nasal insufflation. Enteral administration is alsopossible. For such administration, the product should be formulated intoan appropriate capsule or elixir for oral administration, or into asuppository for rectal administration.

The foregoing exemplary administration modes will likely require thatthe product be formulated into an appropriate carrier, includingointments, gels, suppositories. Appropriate formulations are well knownto persons skilled in the art.

Dosage of the product will vary, depending upon the potency andtherapeutic index of the particular polypeptide selected.

A therapeutic composition for use in the treatment method can includethe product in a sterile injectable solution, the polypeptide in an oraldelivery vehicle, the product in an aerosol suitable for nasaladministration, or the product in a nebulized form, all preparedaccording to well known methods. Such compositions comprise atherapeutically effective amount of the compound, and a pharmaceuticallyacceptable carrier or excipient. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The product of the invention may also be usedto modulate endothelial differentiation and proliferation as well as tomodulate apoptosis either ex vivo or in vitro, for example, in cellcultures.

EXAMPLE III Anti-Variant Antibodies

A. Synthesis

In still another aspect of the invention, the purified variants productsare used to produce anti-variant antibodies which have diagnostic andtherapeutic uses related to the activity, distribution, and expressionof the CD40R variants products.

Antibodies to the CD40R variant may be generated by methods well knownin the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragmentsand fragments produced by an Fab expression library. Antibodies, i.e.,those which inhibit dimer formation, are especially preferred fortherapeutic use.

A fragment of the CD40R variants products for antibody induction is notrequired to feature biological activity but has to feature immunologicalactivity; however, the protein fragment or oligopeptide must beantigenic. Peptides used to induce specific antibodies may have an aminoacid sequence consisting of at least five amino acids, preferably atleast 10 amino acids of the sequences specified in SEQ ID NO:7 to SEQ IDNO:12. Preferably they should mimic a portion of the amino acid sequenceof the natural protein and may contain the entire amino acid sequence ofa small, naturally occurring molecule. Short stretches of CD40R variantsproteins amino acids may be fused with those of another protein such askeyhole limpet hemocyanin and antibody produced against the chimericmolecule. Procedures well known in the art can be used for theproduction of antibodies to CD40R variants products.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc may be immunized by injection with CD40Rvariants products or any portion, fragment or oligopeptide which retainsimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants includebut are not limited to Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacteriumparvum are potentially useful human adjuvants.

Monoclonal antibodies to CD40R variants protein may be prepared usingany technique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Koehler and Milstein(Nature 256:495-497, (1975)), the human B-cell hybridoma technique(Kosbor et al., Immunol. Today 4:72, (1983); Cote et al., Proc. Natl.Acad. Sci. 80:2026-2030, (1983)) and the EBV-hybridoma technique (Cole,et al., Mol. Cell Biol. 62:109-120, (1984)).

Techniques developed for the production of “chimeric antibodies”, thesplicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate antigen specificity and biological activitycan also be used (Morrison et al., Proc. Natl. Acad. Sci. 81:6851-6855,(1984); Neuberger et al., Nature 312:604-608, (1984); Takeda et al.,Nature 314:452454, (1985)). Alternatively, techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778) can beadapted to produce single-chain antibodies specific for the variantprotein.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al. (Proc. Natl. Acad. Sci. 86:3833-3837, 1989)), and WinterG and Milstein C., (Nature 349:293-299, (1991)).

Antibody fragments which contain specific binding sites for the CD40Rvariant protein may also be generated. For example, such fragmentsinclude, but are not limited to, the F(ab′)₂ fragments which can beproduced by pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse W. D. et al., Science256:1275-1281, (1989)).

B. Diagnostic Applications of Antibodies

A variety of protocols for competitive binding or immunoradiometricassays using either polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Such immunoassays typicallyinvolve the formation of complexes between the CD40R variants productsand its specific antibody and the measurement of complex formation. Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two noninterfering epitopes on a specific variant product ispreferred, but a competitive binding assay may also be employed. Theseassays are described in Maddox D. E., et al., (J. Exp. Med. 158:1211,(1983)).

Antibodies which specifically bind the CD40R variant product are usefulfor the diagnosis of conditions or diseases characterized by expressionof the novel CD40R variants of the invention (where normally it is notexpressed) by over or under expression of CD40R variants as well as fordetection of diseases in which the proportion between the amount of theCD40R variants of the invention and the original CD40R sequence fromwhich it varied is altered. Alternatively, such antibodies may be usedin assays to monitor patients being treated with CD40R variantsproducts. Diagnostic assays for variants proteins include methodsutilizing the antibody and a label to detect variants products in humanbody fluids or extracts of cells or tissues. The products and antibodiesof the present invention may be used with or without modification.Frequently, the proteins and antibodies will be labeled by joining them,either covalently or noncovalently, with a reporter molecule A widevariety of reporter molecules are known in the art.

A variety of protocols for measuring the CD40R variants products, usingeither polyclonal or monoclonal antibodies specific for the respectiveprotein are known in the art. Examples include enzyme-lied immunosorbentassay (ELISA), radioimmunoassay (RIA), and fluorescent activated cellsorting (FACS). As noted above, a two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson CD40R variants products is preferred, but a competitive binding assaymay be employed. These assays are described, among other places, inMaddox, et al. (supra). Such protocols provide a basis for diagnosingaltered or abnormal levels of CD40R variants products expression. Normalor standard values for CD40R variants products expression areestablished by combining body fluids or cell extracts taken from normalsubjects, preferably human, with antibodies to CD40R variants productsunder conditions suitable for complex formation which are well known inthe art. The amount of standard complex formation may be quantified byvarious methods, preferably by photometric methods. Then, standardvalues obtained from normal samples may be compared with values obtainedfrom samples from subjects potentially affected by disease. Deviationbetween standard and subject values establishes the presence of diseasestate.

The antibody assays are useful to determine the level of CD40R variantsproducts present in a body fluid sample, in order to determine whetherit is being expressed at all, whether it is being overexpressed orunderexpressed in the tissue, or as an indication of how CD40R variantslevels of variable products are responding to drug treatment.

C. Therapeutic Uses of Antibodies

In addition to their diagnostic use the antibodies may have atherapeutical utility in blocking or decreasing the activity of theCD40R variant product in pathological conditions where beneficial effectcan be achieved by such a decrease.

The antibody employed is preferably a humanized monoclonal antibody, ora human Mab produced by known globulin-gene library methods. Theantibody is administered typically as a sterile solution by IVinjection, although other parenteral routes may be suitable. Typically,the antibody is administered in an amount between about 1-15 mg/kg bodyweight of the subject. Treatment is continued, e.g., with dosing every1-7 days, until a therapeutic improvement is seen.

Although the invention has been described with reference to specificmethods and embodiments, it is appreciated that various modificationsand changes may be made without departing from the invention.

1. An isolated polypeptide comprising the amino acid sequence set forthin SEQ ID NO:
 7. 2. A pharmaceutical composition comprising thepolypeptide of claim 1 and a pharmaceutically acceptable carrier.